CN115216492B - Preparation method and application of mouse primary glioma model - Google Patents
Preparation method and application of mouse primary glioma model Download PDFInfo
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Abstract
The invention discloses a preparation method and application of a mouse primary glioma model, comprising the following steps: 1) Constructing PB-CAG-MCS-IRES-EGFP plasmid, 2) constructing PB-CAG-Pdgfa-HA-IRES-EGFP plasmid, inserting Pdgfa gene into a multiple cloning site of the PB-CAG-MCS-IRES-EGFP plasmid, and expressing PDGFA protein carrying HA tag and green fluorescent protein EGFP for tracing; 3) And (3) preparing a primary glioma mouse model to obtain the primary glioma mouse model. The invention provides an important research model for the related research such as glioma occurrence, treatment and the like.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method and application of a mouse primary glioma model.
Background
Gliomas (gliomas) are common tumors of the central nervous system. Glioblastoma (GBM) is a more malignant tumor of the nervous system. Glioblastoma GBM can be classified into a neural anterior type (PN), a Mesenchymal (MES) type (MES), a Classical (CL) type (NL) and a neural type (NL) according to characteristics such as gene expression. Different tumor subtypes are often associated with different genetic mutations. For example, over 90% of negative CL-GBMs have enhanced EGFR activity and CDKN2A deletions, MES-GBMs are often associated with NF1/PTEN gene mutations, whereas PDGFRA amplification and IDH1 gene mutations can be observed in the vast majority of PN-GBMs. The current research model of glioma mainly comprises glioma cells cultured in vitro, such as A172, U-251 and the like; or by inoculating in vitro cultured tumor cells into mouse brain. However, these models can only study the progress after tumor formation, the therapeutic effect and other problems, but cannot study the problem of tumor occurrence, and the tumor cells in the cell line state often have great differences from the primary glioma in vivo. Thus, the primary glioma model is an indispensable tool for glioma studies and drug screening.
The preparation of the primary glioma model at present mainly depends on the use of a plurality of genetically modified mice, and the preparation difficulty is high. The common methods are as follows: (1) Pten is combined by means of a tool mouse expressing Cre recombinase such as NG2-creER, hGFAP-creER, nestin-Cre and the like fx/fx Or p53 fx/fx Mouse strains such as LSL-EGFRviii, etc., conditional knockdown of p53, nf1, pten, etc. genes or activation of EGFR, PDGFRa signaling pathway in oligodendrocyte precursor cells (oligodendrocyte progenitor cells, OPCs), neural precursor cells; (2) Replication and infection of avian viruses in mouse nerve cells are realized by injecting an avian virus RCAS plasmid carrying an exogenous gene into the brain of a mouse by means of an Olig2-TVA, hGFAP-TVA and Nestin-TVA tool mouse expressing an avian virus receptor A (avian tumor virus receptor A, TVA), mediating the continuous expression of the exogenous gene, and finally inducing glioma; (3) Inactivation of these cancer suppressor genes was achieved by electrotransfection of plasmids expressing small guide RNAs for the p53, nf1 and Pten genes by means of a transgenic mouse expressing Cas9, thereby inducing primary gliomas in the mice; (4) Transposon systems have also been recently adopted to induce gliomas by transposing and integrating exogenous genes into brain nerve cells of specific genetically modified mice for the purpose of activating or knocking out specific genes. However, these primary glioma models are all based on genetically modified mice and are therefore limited in practice by the raw materials. Therefore, there is a need to develop a primary glioma model that can be achieved in normal wild-type mice.
The piggyBac transposon system has also been shown to be effective in achieving stable expression of exogenous genes in mouse cells, and has higher efficiency than other transposon systems, and is an ideal method for expressing exogenous genes in mouse tissues or performing gene knockout. Studies have shown that activation of persistent expression of the Pdgfa gene alone induces the aforementioned PN-GBM-like gliomas in the mouse brain.
Disclosure of Invention
The invention provides a preparation method and application of a mouse primary glioma model, which are used for solving the problems in the prior art.
The scheme of the invention is as follows:
a method for preparing a mouse primary glioma model, comprising the following steps:
1) Constructing a PB-CAG-MCS-IRES-EGFP plasmid, wherein the PB-CAG-MCS-IRES-EGFP plasmid comprises a promoter and a downstream IRES-EGFP sequence, and a multiple cloning site is added between the promoter and the IRES-EGFP sequence;
2) Constructing PB-CAG-Pdgfa-HA-IRES-EGFP plasmid, inserting Pdgfa gene into a multiple cloning site of the PB-CAG-MCS-IRES-EGFP plasmid, and expressing a PDGFA protein carrying an HA tag and a green fluorescent protein EGFP for tracing;
3) Preparation of a Primary glioma mouse model A primary glioma mouse model was obtained by inducing mice by co-using plasmid PB-CAG-Pdgfa-HA-IRES-EGFP with plasmid pCAGGS-piggyBac Transposase expressing a transposase.
As a preferred embodiment, the promoter is a CAG promoter or other promoters useful to mammalian cells.
As a preferable technical scheme, the PB-CAG-Pdgfa-HA-IRES-EGFP plasmid is integrated into a cell genome in the presence of piggyBac transposase, so that stable expression of a target gene in a transferred cell and a progeny cell thereof is realized.
The invention also discloses a preparation method of the mouse primary glioma model, which uses piggyBac transposon plasmid PB-CAG-MCS-IRES-EGFP, wherein the nucleotide sequence of the PB-CAG-MCS-IRES-EGFP is shown as SEQ ID No. 1.
The invention also discloses application of the preparation method of the primary glioma model of the mouse, and provides an important research model for glioma occurrence and treatment research.
Due to the adoption of the technical scheme, the preparation method of the mouse primary glioma model comprises the following steps of: 1) Constructing a PB-CAG-MCS-IRES-EGFP plasmid, wherein the PB-CAG-MCS-IRES-EGFP plasmid comprises a promoter and a downstream IRES-EGFP sequence, and a multiple cloning site is added between the promoter and the IRES-EGFP sequence; 2) Constructing PB-CAG-Pdgfa-HA-IRES-EGFP plasmid, inserting Pdgfa gene into a multiple cloning site of the PB-CAG-MCS-IRES-EGFP plasmid, and expressing a PDGFA protein carrying an HA tag and a green fluorescent protein EGFP for tracing; 3) Preparation of a Primary glioma mouse model A primary glioma mouse model was obtained by inducing mice by co-using plasmid PB-CAG-Pdgfa-HA-IRES-EGFP with plasmid pCAGGS-piggyBac Transposase expressing a transposase.
The invention has the advantages that:
the invention constructs a piggyBac transposon plasmid carrying EGFP, PB-CAG-Pdgfa-HA-IRES-EGFP plasmid is obtained by inserting Pdgfa genes into the plasmid, the plasmid is transferred into the mouse cerebral cortex of embryo period 14.5 (E14.5) by a mouse embryo plasmid electrotransformation technology, and primary glioma is induced after the birth of the mouse. With the plasmid system constructed and the described method, primary gliomas can be induced in normal wild-type mouse brains without the need for various genetically modified mice. The invention provides an important research model for the related research such as glioma occurrence, treatment and the like.
Drawings
FIG. 1 is a phenotype chart of a primary glioma model of a Pdgfa-induced mouse according to an embodiment of the present invention;
FIG. 2 is a diagram showing the detection of the expression of each marker in the primary glioma by immunohistochemistry and in situ hybridization according to the embodiment of the present invention;
FIG. 3 shows a plasmid map of PB-CAG-MCS-IRES-EGFP of the present invention.
Detailed Description
In order to overcome the defects, the invention provides a preparation method and application of a mouse primary glioma model, and aims to solve the problems in the background technology.
A method for preparing a mouse primary glioma model, comprising the following steps:
1) Constructing a PB-CAG-MCS-IRES-EGFP plasmid, wherein the PB-CAG-MCS-IRES-EGFP plasmid comprises a promoter and a downstream IRES-EGFP sequence, and a multiple cloning site is added between the promoter and the IRES-EGFP sequence;
2) Constructing PB-CAG-Pdgfa-HA-IRES-EGFP plasmid, inserting Pdgfa gene into a multiple cloning site of the PB-CAG-MCS-IRES-EGFP plasmid, and expressing a PDGFA protein carrying an HA tag and a green fluorescent protein EGFP for tracing;
3) Preparation of a Primary glioma mouse model A primary glioma mouse model was obtained by inducing mice by co-using plasmid PB-CAG-Pdgfa-HA-IRES-EGFP with plasmid pCAGGS-piggyBac Transposase expressing a transposase.
The promoter is a CAG promoter or other promoters useful for mammalian cells.
The PB-CAG-Pdgfa-HA-IRES-EGFP plasmid is integrated into a cell genome in the presence of piggyBac transposase, so that stable expression of a target gene in a transferred cell and a progeny cell thereof is realized.
The invention also discloses a preparation method of the mouse primary glioma model, which uses piggyBac transposon plasmid PB-CAG-MCS-IRES-EGFP, wherein the nucleotide sequence of the PB-CAG-MCS-IRES-EGFP is shown as SEQ ID No. 1. The plasmid contains a CAG promoter and IRES-EGFP sequence at the downstream of the CAG promoter, and a multiple cloning site is added between the CAG promoter and the IRES sequence for inserting exogenous genes. The plasmid contains a nucleic acid sequence required by piggyBac transposase mediated transposition, and can realize stable expression of a target gene in a transferred cell and a progeny cell thereof.
The plasmid pCAGGS-piggyBac Transposase expressing piggyBac transposase was designated pCAGGS-PBase.
The plasmid PB-CAG-Pdgfa-HA-IRES-EGFP was co-electrotransferred to embryonic mouse cerebral cortex by mouse intrauterine embryo electrotransfer (in ovo electroporation, IOE) with plasmid pCAGGS-piggyBac Transposase expressing a transposase, thereby inducing the mouse to produce a primary glioma post-natally.
Based on a transposon gene delivery system, the invention realizes the continuous overexpression of Pdgfa in the brain of the mouse by an embryo electrotransformation method, thereby obtaining a primary glioma model of the mouse. Under the technical conditions of using the transposon system, it is within the scope of the claims of the present invention to replace the CAG promoter of the present invention with promoters usable for other mammalian cells, or to adjust the development period of the embryo-electrotransferred mouse, such as 12 days (E12) to 1 week (P7) after birth in the embryo period.
The invention also discloses application of the preparation method of the primary glioma model of the mouse, and provides an important research model for glioma occurrence and treatment research.
The invention is further described in connection with the following embodiments in order to make the technical means, the creation features, the achievement of the purpose and the effect of the invention easy to understand.
Examples:
construction of PB-CAG-MCS-IRES-EGFP plasmid:
first, the CAG-MCS-IRES-EGFP fragment needs to be obtained. The fragment comprises two fragments of a CAG promoter and IRES-EGFP. By the technique of overlap extension PCR, two DNA fragments can be spliced together. Wherein the CAG promoter fragment is derived from pCAGGS-mCherry plasmid and the IRES-EGFP fragment is derived from pCIG 2-Flag-mNaak 1 plasmid. The primers were designed as follows:
CAG-IRES-F:
5-gccaccatgtgtacagaactcgagtttaaatcggatccgatccgcccctctccctcccccccccctaacgttact-3
CAG-IRES-R:
5-ctcgagttctgtacacatggtggctgacatctgatgatggctagccccgggcccgcggtaccgtcgact-3
CAG-F:5-GGACTAGTtattaatagtaatcaattacggggtcattagttcatagcccat-3EGFP-R:
5-GCACGCGTCGACtgtaccgctcatctgttagtggtgcctagattacttgtagagctcgtccatgcc-3
PCR-1# amplification was performed using primers CAG-F and CAG-IRES-R as PCR primers, the template being plasmid pCAGGS-mCherry (Addgene, # 41583), and the target product was approximately 1.7kb. PCR-2# amplification was performed using primers CAG-IRES-F and EGFP-R as primers, and the template was plasmid pCIG2-Flag-mNuak1 (Addgene, # 168507), with the target product of about 1.4kb. The 2 PCRs were each 30uL system, 5 cycles. The enzyme was PrimeSTAR GXL DNA Polymerase (#R050Q) from TAKARA, and the amplification system was referred to the DNA polymerase instructions. After the PCR-1# and the PCR-2# are finished, a third round of PCR reaction is carried out by taking the two PCR products as templates (the using amount is 1uL/30uL system) and taking CAG-F and EGFP-R as primers, namely the PCR-3# is obtained. The PCR reaction was carried out in the same manner as above, but the amplification was 60uL, the cycle number was set to 30, and the target fragment size was about 3.2kb. After the amplification, the PCR-3# product was subjected to 1% agarose gel electrophoresis, followed by purification of the target fragment (50 uL of water eluted product) by a gel recovery kit. The DNA fragment is CAG-MCS-IRES-EGFP. The target fragment was digested with SpeI and SalI (enzyme purchased from Thermo, # FD1254, # FD 0644), 50uL of the system, 43uL of the gel recovery product, buffer 5uL, 1uL of each of the two endonucleases, and incubated overnight at 37 ℃. The DNA Clean-Up kit recovers the digested PCR product to 30uL of deionized water for later use.
Thereafter, it is necessary to obtain a plasmid fragment which can be recognized by the transposase. The primers were designed as follows:
PB-F:5-GCACGCGTCGACaatcaacctctggattacaaaatttgtg-3
PB-R:5-GGACTAGTggccttggaggccttttccccgtatccccccag-3
primers PB-F and PB-R were used as PCR, and the template was plasmid PB-CMV-MCS-EF1 alpha-GFP-Puro (SBI, #PB513B-1). The PCR product fragment was about 4.9kb in size and the reaction system was the same as before. After agarose gel electrophoresis of the PCR product, the PCR product was purified and recovered, and then digested with SpeI and SalI. The subsequent fragment recovery method is the same as above. Ligating the double digested fragment with the CAG-MCS-IRES-EGFP fragment: 3.2kb double enzyme fragment 3uL, CAG-MCS-IRE-EGFP double enzyme fragment 5.5uL,Ligase Buffer 1uL,T4 Ligase 0.5uL. E.coli DH5a competent cells were transformed by ligation for 30min at room temperature. The following day, monoclonal colonies were picked for sequencing. Clones sequenced correctly can be inoculated and cultured and plasmids extracted. Namely, PB-CAG-MCS-IRES-EGFP plasmid is obtained. The plasmid introduced a multiple cloning site between the CAG promoter and IRES, containing NheI and BsrGI restriction enzyme recognition sites, for subsequent insertion of the foreign gene fragment (fig. 3).
Construction of PB-CAG-Pdgfa-HA-IRES-EGFP plasmid:
in order to obtain the Pdgfa fragment, primers were designed as follows:
Pdgfa-F:5-GCTCTAGAGCCACCatgaggacctgggcttgcctgctg-3
Pdgfra-R:
5-GCGCTGGTACCttatgcataatcaggcacatcgtaaggatacctcacatctgtctcctcctcccg-3
the PCR method, the DNA fragment recovery method and the DNA fragment double digestion and purification method are the same as described above. The template used in PCR is cDNA obtained by reverse transcription of total RNA extracted from mouse brain tissue 2 weeks after birth, and the target fragment size is about 630bp.
Next, nheI and BsrGI double cleavage was performed on vector PB-CAG-MCS-IRES-EGFP, i.e.plasmid 5ug,50uL system, and endonucleases were incubated at 37℃overnight for each 2.5 uL. The target fragment of about 8kb (50 uL of water eluted product) was purified by 1% agarose gel electrophoresis using a gel recovery kit. Ligating the fragment to the aforementioned Pdgfa-HA fragment: 1uL of vector fragment, 5uL of Pdgfa-HA double enzyme fragment, deionized water 2.5uL,Ligase Buffer 1uL,T4 Ligase 0.5uL. E.coli DH5a competent cells were transformed by ligation for 30min at room temperature. The following day, monoclonal colonies were picked for sequencing. Clones sequenced correctly can be inoculated and cultured and plasmids extracted. Namely, PB-CAG-Pdgfa-HA-IRES-EGFP plasmid is obtained, and the plasmid can simultaneously express green fluorescent protein EGFP and HA-tag-carrying PDGFA protein under the drive of a CAG promoter.
3. Preparation of primary glioma mouse model:
plasmid PB-CAG-Pdgfa-HA-IRES-EGFP was extracted from E.coli by endotoxin-removing plasmid extraction kit, and dissolved in endotoxin-free deionized water to a concentration of 5000 ng/. Mu.l. Female and male adult mice were placed in the same cage in the evening, separated the next morning, and scored as embryonic day 0.5 (E0.5) to noon. E14.5 pregnant mice were taken for mouse embryo plasmid electrotransformation. Detailed experimental procedures were performed as reported in the previous literature. The general flow is as follows: sterilizing the surface of abdomen of pregnant mice after anesthesia, cutting off abdominal hair, opening abdominal cavity, pulling out uterus of mice containing embryo, injecting 2-3uL plasmid mixed with FastGreen dye with final concentration of 0.01% into ventricles of embryo side by microinjection instrument, placing circular electrode with diameter of 6cm on two sides of embryo brain, applying 45V pulse voltage (50 ms each time, 950ms each time, 5 times of continuous electric shock); the uterus was carefully moved back to the mouse uterus, the wound was sutured, sterilized and left at 37 ℃ to maintain body temperature until the mouse wakes up. The mice pups were born approximately after 4-5 days, and glioma formation was examined when postnatal mice survived to 1 month of age. Control mice were electrotransformed with plasmid PB-CAG-MCS-IRES-EGFP.
The results showed that after one month of birth, the head of the mice had malformations, the mobility of the mice was severely affected, and the body type was smaller than that of the mice in the control group (FIGS. 1A-C). The brain tissue was subjected to a slice analysis after dissection, and the results showed that: the tumor site volume of the mouse cerebral cortex has been equivalent to the size of normal brain tissue, and in situ hybridization experiments against Pdgfa mRNA confirm that there is extensive high expression of the Pdgfa gene in the tumor-forming region (FIGS. 1D, E). Immunofluorescence and immunohistochemical staining showed that cells at tumor sites highly expressed markers of PN-GBM such as OLIG2, PDGFRA, SOX10 (FIGS. 2A, B, A ', B'). Meanwhile, in the tumor area, gfap+/glass+ astrocytes were absent (fig. 2D), few neun+ neurons were present (fig. 2C), but there were a number of iba1+/cd68+ activated microglia (fig. 2e, D). These results indicate that the method was used to successfully construct a mouse primary glioma model. The foregoing has shown and described the basic principles, main features and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
<110> Zhejiang Orsei Biotechnology Co., ltd
<120> preparation method and application of mouse primary glioma model
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8070
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat 60
tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga 120
tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca 180
acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct 240
aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc 300
cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag 360
cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca 420
cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca 480
actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg 540
gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta 600
aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg gaggacgggc 660
agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg ctcgacacgc 720
tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg tacgttaaag 780
ataatcatgc gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag gtttatttat 840
taatttgaat agatattaag ttttattata tttacactta catactaata ataaattcaa 900
caaacaattt atttatgttt atttatttat taaaaaaaaa caaaaactca aaatttcttc 960
tataaagtaa caaaactttt atgagggaca gccccccccc aaagccccca gggatgtaat 1020
tacgtccctc ccccgctagg gggcagcagc gagccgcccg gggctccgct ccggtccggc 1080
gctccccccg catccccgag ccggcagcgt gcggggacag cccgggcacg gggaaggtgg 1140
cacgggatcg ctttcctctg aacgcttctc gctgctcttt gagcctgcag acacctgggg 1200
ggatacgggg aaaaggcctc caaggccact agttattaat agtaatcaat tacggggtca 1260
ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct 1320
ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta 1380
acgccaatag ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac 1440
ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 1500
aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag 1560
tacatctacg tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt 1620
cactctcccc atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt 1680
attttgtgca gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg 1740
gcgaggggcg gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc 1800
tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg 1860
cgcggcgggc gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc 1920
gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc 1980
cttctcctcc gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc 2040
tgcgtgaaag ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg 2100
ggtgcgtgcg tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct 2160
gtgagcgctg cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc 2220
ggccgggggc ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg 2280
ggtgtgtgcg tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc 2340
cctgcacccc cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtac 2400
ggggcgtggc gcggggctcg ccgtgccggg cggggggtgc cggcaggtgg gggtgccggg 2460
cggggcgggg ccgcctcggg cctgggaggg ctcgggggag gggcgcggcg gcccccggag 2520
cgccggcggc tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga 2580
gagggcgcag ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg 2640
ccgcaccccc tctagcgggc gcggggcgaa gcggtgcggc gccggcagga aggaaatggg 2700
cggggagggc cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg 2760
ctgtccgcgg ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg 2820
gcgtgtgacc ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct 2880
acagctcctg ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattcct 2940
cgagatctcg agctcaagct tcgaattctg cagtcgacgg taccgcgggc ccggggctag 3000
ccatcatcag atgtcagcca ccatgtgtac agaactcgag tttaaatcgg atccgatccg 3060
cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg 3120
tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg 3180
gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg 3240
aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca 3300
aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct 3360
ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca 3420
cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa 3480
ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggta 3540
cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg 3600
ggacgtggtt ttcctttgaa aaacacgatg ataatatggc cacaaccatg gtgagcaagg 3660
gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg 3720
gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc 3780
tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc 3840
tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct 3900
tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg 3960
gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg 4020
agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca 4080
actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga 4140
acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc 4200
agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc 4260
agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg 4320
tgaccgccgc cgggatcact ctcggcatgg acgagctcta caagtaatct aggcaccact 4380
aacagatgag cggtacagtc gacaatcaac ctctggatta caaaatttgt gaaagattga 4440
ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt 4500
tgtatcagtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac 4560
aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat 4620
caatgtatct tatcatgtct ggaattgact caaatgatgt caattagtct atcagaagct 4680
atctggtctc ccttccgggg gacaagacat ccctgtttaa tatttaaaca gcagtgttcc 4740
caaactgggt tcttatatcc cttgctctgg tcaaccaggt tgcagggttt cctgtcctca 4800
caggaacgaa gtccctaaag aaacagtggc agccaggttt agccccggaa ttgactggat 4860
tcctttttta gggcccattg gtatggcttt ttccccgtat ccccccaggt gtctgcaggc 4920
tcaaagagca gcgagaagcg ttcagaggaa agcgatcccg tgccaccttc cccgtgcccg 4980
ggctgtcccc gcacgctgcc ggctcgggga tgcgggggga gcgccggacc ggagcggagc 5040
cccgggcggc tcgctgctgc cccctagcgg gggagggacg taattacatc cctgggggct 5100
ttgggggggg gctgtccctg atatctataa caagaaaata tatatataat aagttatcac 5160
gtaagtagaa catgaaataa caatataatt atcgtatgag ttaaatctta aaagtcacgt 5220
aaaagataat catgcgtcat tttgactcac gcggtcgtta tagttcaaaa tcagtgacac 5280
ttaccgcatt gacaagcacg cctcacggga gctccaagcg gcgactgaga tgtcctaaat 5340
gcacagcgac ggattcgcgc tatttagaaa gagagagcaa tatttcaaga atgcatgcgt 5400
caattttacg cagactatct ttctagggtt aatctagctg catcaggatc atatcgtcgg 5460
gtcttttttc cggctcagtc atcgcccaag ctggcgctat ctgggcatcg gggaggaaga 5520
agcccgtgcc ttttcccgcg aggttgaagc ggcatggaaa gagtttgccg aggatgactg 5580
ctgctgcatt gacgttgagc gaaaacgcac gtttaccatg atgattcggg aaggtgtggc 5640
catgcacgcc tttaacggtg aactgttcgt tcaggccacc tgggatacca gttcgtcgcg 5700
gcttttccgg acacagttcc ggatggtcag cccgaagcgc atcagcaacc cgaacaatac 5760
cggcgacagc cggaactgcc gtgccggtgt gcagattaat gacagcggtg cggcgctggg 5820
atattacgtc agcgaggacg ggtatcctgg ctggatgccg cagaaatgga catggatacc 5880
ccgtgagtta cccggcgggc gcgcttggcg taatcatggt catagctgtt tcctgtgtga 5940
aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc 6000
tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc 6060
cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 6120
ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 6180
cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 6240
ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 6300
aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 6360
cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 6420
cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 6480
gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 6540
tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 6600
cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 6660
ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 6720
gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc 6780
gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 6840
accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 6900
ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 6960
tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 7020
aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 7080
taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 7140
gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 7200
agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac 7260
cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 7320
tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 7380
gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 7440
agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 7500
gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 7560
atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 7620
gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 7680
tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 7740
atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 7800
agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 7860
gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 7920
cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 7980
tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 8040
ccgcgcacat ttccccgaaa agtgccacct 8070
<210> 2
<211> 615
<212> DNA
<213> Mus musculus
<400> 2
atgaggacct gggcttgcct gctgctcctc ggctgcggat acctcgccca tgccctggcc 60
gaggaagccg agataccccg ggagttgatc gagcggctgg ctcgaagtca gatccacagc 120
atccgggacc tccagcgact cttggagata gactccgtag gggctgagga tgccttggag 180
acaagtctga gagcccatgg gtcccatgcc attaaccatg tgcccgagaa gcggcctgtg 240
cccattcgca ggaagagaag tattgaggaa gccattcctg cagtttgcaa gaccaggacg 300
gtcatttacg agatacctcg gagccaggtg gaccccacat cggccaactt cctgatctgg 360
cccccatgtg tggaggtgaa gcgctgcact ggctgttgta acaccagcag cgtcaagtgc 420
cagccttcac gggtccacca ccgcagtgtc aaggtggcca aagtggagta tgtcaggaag 480
aagccaaaat tgaaagaggt ccaggtgagg ttagaggaac acctggagtg tgcatgtgcg 540
acctccaacc tgaacccaga ccatcgggag gaggagacag atgtgaggta tccttacgat 600
gtgcctgatt atgca 615
Claims (2)
1. A method for preparing a primary glioma model of a mouse, which is characterized by comprising the following steps:
1) Constructing a PB-CAG-MCS-IRES-EGFP plasmid, wherein the PB-CAG-MCS-IRES-EGFP plasmid comprises a promoter and a downstream IRES-EGFP sequence, a multiple cloning site is added between the promoter and the IRES-EGFP sequence, and the nucleotide sequence of the PB-CAG-MCS-IRES-EGFP is shown as SEQ ID No. 1;
2) Constructing PB-CAG-Pdgfa-HA-IRES-EGFP plasmid, inserting Pdgfa gene into a multiple cloning site of the PB-CAG-MCS-IRES-EGFP plasmid, and expressing a PDGFA protein carrying an HA tag and a green fluorescent protein EGFP for tracing;
3) Preparing a primary glioma mouse model, namely, using a plasmid PB-CAG-Pdgfa-HA-IRES-EGFP together with a plasmid pCAGGS-piggyBac Transposase for expressing transposase to induce a mouse, so as to obtain the primary glioma mouse model; the PB-CAG-Pdgfa-HA-IRES-EGFP plasmid is integrated into a cell genome in the presence of piggyBac transposase, so that stable expression of a target gene in a transferred cell and a progeny cell thereof is realized.
2. Use of the method for preparing a mouse primary glioma model according to claim 1, characterized in that: provides a research model for the occurrence and treatment research of glioma.
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| US9750826B2 (en) * | 2012-06-21 | 2017-09-05 | Samsung Life Public Welfare Foundation | Method for preparing patient-specific glioblastoma animal model, and uses thereof |
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| US20200384022A1 (en) * | 2017-11-13 | 2020-12-10 | The Broad Institute, Inc. | Methods and compositions for targeting developmental and oncogenic programs in h3k27m gliomas |
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